Sanger Sequencing

Genomics DNA Sequencing
1

Hi,

I have designed primers that bind to conserved regions, so that variable regions in-between the conserved regions get amplified. My main aim is to detect the microbial diversity in a given sample by amplifying the variable regions of different microbes. I used to amplify variable regions and get a 340 bp product and with this product i used to clone it by topo vector and later on used to sequence each single clone by sanger sequencing and the sequences were very fine. But when i gave the 340 bp PCR product directly for sanger sequencing there was presence of muliple peaks.so iam stuck here.Why my sequencing is not working.what is the problem? i give exosap treatment before sequencing. Some one please help me out what went wrong in my experiment or is it not possible to get sequences of variable region amplicon? please help me out with your valuable suggestion

2

Laura

The issue you’re encountering with multiple peaks in your Sanger sequencing chromatogram likely stems from the heterogeneity of your 340 bp PCR product. When you amplify the conserved regions flanking variable regions from a mixed microbial community, the resulting PCR product is a mixture of amplicons from different microbial species, each with a potentially different sequence in the variable region. This mixture causes overlapping peaks in Sanger sequencing, making it difficult to resolve individual sequences.

You can modify the conditions of your PCR to minimize amplification biases that could preferentially amplify some variants over others.

Also, you can try to sub-cloning portions of your PCR product or use single-molecule sequencing to capture a broader range of the diversity

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